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The Nuclear Pore Complex pp 3–34Cite as

Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2502))

Abstract

Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker’s yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.

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Acknowledgments

This work was supported by National Institutes of Health grants U54 GM103511, R01 GM112108, P41 GM109824 and U54 DK107981. We thank all members of the Laboratory of Cellular and Structural Biology (Rout lab) and the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry (headed by Prof. Brian Chait), especially Wenzhu Zhang and Erica Jacobs, for their assistance with the development and testing of this protocol as well as for their feedback and helpful discussions. We thank Azraa S. Chaudhury for her help with protocol optimization. We gratefully acknowledge Kunihiro Uryu and the EMRC Resource Center (Rockefeller University) for teaching and supporting our negative stain electron microscopy analysis. We are grateful to Dan Simon for support, invaluable discussions, and critical reading of the manuscript.

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Authors and Affiliations

  1. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA

    Ilona Nudelman, Javier Fernandez-Martinez & Michael P. Rout

  2. Fisher Drug Discovery Resource Center (DDRC), The Rockefeller University, New York, NY, USA

    Ilona Nudelman

  3. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Javier Fernandez-Martinez

  4. Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country, Leioa, Bizkaia, Spain

    Javier Fernandez-Martinez

Authors
  1. Ilona Nudelman
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  2. Javier Fernandez-Martinez
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  3. Michael P. Rout
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Corresponding author

Correspondence to Michael P. Rout .

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Editors and Affiliations

  1. Department of Biosciences, Durham University, Durham, UK

    Martin W. Goldberg

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Nudelman, I., Fernandez-Martinez, J., Rout, M.P. (2022). Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes. In: Goldberg, M.W. (eds) The Nuclear Pore Complex. Methods in Molecular Biology, vol 2502. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2337-4_1

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  • DOI: https://doi.org/10.1007/978-1-0716-2337-4_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2336-7

  • Online ISBN: 978-1-0716-2337-4

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